Dyes and methods of detection of nucleic acid in immature red blood cells

ABSTRACT

The dyes of the present invention are useful for many purposes that include markers or tags for detecting the presence of a molecule or compound to which they are bound. The dyes may be either red-excitable or blue-excitable. The dyes of the invention are particularly well suited for staining of nucleic acids. For example, these dyes are particularly suitable for staining of RNA in reticulocytes. In another exemplary application, these dyes are suitable for staining DNA in nucleated red blood cells. Typically, when used in staining of nucleic acids, the dyes are formulated into reagent solutions. In addition, the invention provides compositions and methods for facilitating rapid transport of dye molecules through a cell membrane. Such rapid staining requires that a sample be contacted with a dye composition of the invention in the presence of at least one surfactant and optionally, a sulfonic acid or a salt thereof.

REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.09/566,298 filed on May 5, 2000 and having the title “Dyes and Methodsof Reticulocyte Enumeration” now issued as U.S. Pat. No. 6,368,864,issued on Apr. 9, 2002.

FIELD OF THE INVENTION

This invention generally relates to the field of new dyes, dyecompositions, and methods of detection of nucleic acids in red bloodcells by flow cytometry by employing fluorescent, nucleic acid bindingdyes. More particularly, this invention provides a reagent and methodfor enumeration of reticulocytes and nucleated red blood cells.

BACKGROUND OF THE INVENTION

Analysis of red blood cells and enumeration of its immature subpopulations are a valuable components of diagnostic hematology. Forexample, enumeration of reticulocytes, i.e., the immature erythrocytes,in human peripheral blood is useful in the diagnoses of hemorrhage,anemia, monitoring bone marrow transplantation and for monitoringpatients undergoing chemotherapy and other disorders involving bloodcell production [U.S. Pat. No. 5,360,739; H. Shapiro, Practical FlowCytometry, 3^(rd) edit., 1995; Wiley-Liss, New York; Davis et al, (1990)Pathobiol., 58:99-106; Hoy, (1990) Bailliere's Clin. Haemat., 3:977-988;H. J. Tanke, Reticulocytes and Mature Erythrocytes in Flow Cytometry inHaematology (1992) Academic Press Ltd., pp. 75-93]. Becausereticulocytes contain ribonucleic acid (RNA), if stained with RNAbinding excitable dyes, these cells fluoresce when illuminated by alight source of appropriate wavelength. RNA binding dyes have been usedto distinguish reticulocytes from mature red blood cells (RBCs) whichlack RNA.

Distribution of fluorescence intensities of a relatively largereticulocyte population can be determined by flow cytometry in a fastand reliable manner, and different maturation stages of reticulocytes,as reflected by differences in RNA content, can be distinguished.

The use of red-excitable dyes is desirable because such dyes aredetected by excitation with relatively inexpensive diode or HeNe lasers.However, initial efforts in the prior art to employ diode lasers andred-excited dyes for rapid flow cytometric analyses of reticulocyteswere not successful. Yamamoto, U.S. Pat. No. 5,563,070 suggested thatthe addition of large quantities of TO-PRO-3, a red-excitable dye,followed by a 30 minute incubation, stained RNA inside livingreticulocytes. Such a method, however, is not practical for routineanalysis of reticulocytes in clinical laboratories that require highsample throughput because sample preparation time is long and cost pertest is high due to the large amount of dye required to stain eachsample.

A red-excitable dye called Thiazole Blue (TB) has been described in U.S.Pat. No. 4,957,870. However, as described in this patent (U.S. Pat. No.4,957,870), this dye also requires long periods of incubation, of about30 minutes.

Akai et al., U.S. Pat. No. 5,821,127 have also described the preparationof fluorescent dyes which are capable of detecting reticulocytes usinginexpensive detectors via fluorescence in the red region. However thesamples require incubation at elevated temperatures of about 40° C.

More recently, U.S. Pat. No. 5,994,138, described staining reticulocytesvia the use of a red-excitable dye in combination with a detergent andan ionophore at elevated temperatures of about 35° C. However, stainingreticulocytes was not successful when ambient temperatures wereutilized.

Fan et. al (U.S. Pat. No. 5,411,891, U.S. Pat. No. 5,360,739) describesclearly that the specific binding constant between a dye andreticulocyte RNA and the rate of penetration of the dye are differentfor each dye and that it is impossible to predict under what conditionsa particular dye may rapidly penetrate red cell membrane and stainreticulocytes. This was further supported by Akai et. al (U.S. Pat. No.5,821,127).

Thus, there exists a need in the art for dyes, compositions and methodswhich enable rapid staining of intracellular RNA at room temperaturesvia the use of dyes that are excitable in the red region and can useinexpensive and readily available red-illumination instruments. Inaddition there exists a need in the art for dye compositions and methodsthat are applicable not only to red-excitable dyes, but also to dyesexcitable at other wavelengths, such as in the blue wavelength. By doingso, ready and accurate detection of reticulocytes can be accomplishedwithout particular restriction of the excitation wavelength.

In addition to the reticulocytes, another type of immature red bloodcells that are important in clinical diagnostics are the nucleated redblood cells (NRBCs). In contrast to reticulocytes, NRBCs contain DNA.NRBCs normally occur in the bone marrow but not in peripheral blood.However, in certain diseases such as anemia and leukemia, NRBCs alsooccur in peripheral blood. Therefore, it is of clinical importance tomeasure NRBC. Since NRBCs contain DNA, fluorescence detection andenumeration of NRBCs can be possible by staining these cells with dyesthat recognize DNA. Thus, there exists a need in the art for dyes, andcompositions which enable staining of both intracellular DNA and RNA, sothat both NRBCs and reticulocytes can be enumerated via the use of thesame dye employing different methods.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides three groups of novel dyesthat are set forth as Group I, Group II, and Group III with:

Group I having the general formula of:

wherein n is 0, 1, 2, or 3; R₁ is H, alkyl, or an alkoxy group; R₂ isCH₂(CH₂)_(m)OH,

wherein m is 0, 1, 2, or 3; X is O, S, or C(CH₃)₂; R is CH₃, CH(CH₃)₂,CH₂CH₂OH, alkyl, alkylsulfonate, or hydroxyalkyl; and B⁻ is acounteranion.

Group II having the general formula of:

wherein, n is 0, 1, 2, or 3; R1 is H, alkyl, or an alkoxy group; R2 isCH₂(CH₂)_(m)OAc, wherein m is 0, 1, 2, or 3; X is O, S, or C(CH₃)₂; R isCH₃, CH(CH₃)₂, CH₂CH₂OAc, alkyl, alkylsulfonate, or hydroxyalkyl and B−is a counteranion;

and,

Group-III having the following general formula:

wherein n is 0, 1, or 2; R1 is H, alkyl, or an alkoxy group; R2 isCH₂(CH₂)_(m)OH, or CH₃ and wherein m is 0, 1, 2, or 3; X is O, S, orC(CH₃)₂B⁻ is a counter anion, and R3, R4, R5, R6 are varioussubstituents as represented by the formulae for the specific compoundsDye-3 through Dye-6.

In another aspect, the invention provides a reagent containing a dye ofthe invention and a solvent.

In yet another aspect, the invention provides compositions and methodsfor facilitating rapid transport of dye molecules through a cellmembrane. Such rapid staining requires that a sample be contacted with adye composition of the invention in the presence of at least onesurfactant and optionally, a sulfonic acid or a salt thereof.

Other aspects and advantages of this invention will be readily apparentfrom the detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is the comparison of the fluorescence spectra of the dyecompound No. 6 described in example 1, hereinafter called ReticRed1, inPBS solution with or without RNA present in the solution.

FIG. 1B is the comparison of the fluorescence intensity of the dyes ofthis invention and several other dyes studied during this experiment,relative to the fluorescence intensity of a commercial nucleic acid dyeSyto 62 (Molecular Probes, Inc.), all in presence of same amount of RNAin the solution.

FIG. 1C shows the fluorescence intensities of the dyes, ReticRed1through ReticRed6, of this invention in presence of 8 mg/ml RNA comparedto their respective fluorescence intensities when there is no RNApresent in the solution.

FIGS. 2A and 2B show the fluorescence from reticulocytes for an abnormalblood sample which were treated with the dye ReticRed1 in an isotonicsaline solution without the presence of the rapid staining composition,and incubated for two different durations. FIG. 2A shows fluorescencefrom cells treated with ReticRed1 alone and incubated for about 10 min:Total count 42660, Red cells 32841, Reticulocytes 801, calculated reticpercentage 2.4%. FIG. 2B shows fluorescence from cells treated withReticRed1 alone and incubated for about 40 min: Total count 46199, Redcells 39713, Reticulocytes 2310, calculated retic percentage 5.8%. Thereference value for reticulocyte in this sample was approximately 7%.

FIG. 3 shows the fluorescence from reticulocytes in the blood sample,from the same donor that was used in the experiments described in FIGS.2A and 2B, after being treated with a composition of the inventioncontaining the dye ReticRed1 and incubated for about 1 min. Thepercentage of reticulocytes enumerated by this method was 7.3%.

FIG. 4 shows the fluorescence from reticulocytes in an abnormal bloodsample, after being treated with a composition of the inventioncontaining the dye ReticRed1 and incubated for about 1 min. Thepercentage of reticulocytes enumerated by this method was 7.5%. Thereference value for reticulocytes percentage obtained for this donorusing an independent measurement method was 8%. The reagent compositionin this example included Igepal, Dodecyl-β-D-maltoside andp-toluenesulfonic acid monohydrate.

FIG. 5 shows the fluorescence from reticulocytes in the blood samplefrom a normal donor, after being treated with a composition of theinvention containing the dye ReticRed1 and incubated for about 1 min.The percentage of reticulocytes enumerated by this method was 0.82%. Thereagent composition in this example included Igepal,Dodecyl-β-D-maltoside and p-toluenesulfonic acid monohydrate. Thereference value for reticulocyte percentage obtained for this donorusing an independent measurement was 0.95%.

FIG. 6 shows the fluorescence from reticulocytes in an abnormal bloodsample, after being treated with a composition of the inventioncontaining the dye ReticRed1 and incubated for about 1 min. Thepercentage of reticulocytes enumerated by this method was 13.7%. Thereference value for reticulocyte percentage obtained for this donorusing an independent measurement was 13%. The reagent composition inthis embodiment included Igepal, Dodecyl-β-D-maltoside and sodiump-toluenesulfonate.

FIG. 7 shows the fluorescence from reticulocytes in an abnormal bloodsample, after being treated with a composition of the inventioncontaining the dye ReticRed3 and mixed for about 1 sec with no furtherincubation. The percentage of reticulocytes enumerated by this methodwas 13.8%. An independent reference value for reticulocyte percentageobtained for this sample by a conventional Retic-Count™ method wasapproximately 13.3%. The reagent composition in this embodiment includedIgepal®, Dodecyl-β-D-maltoside and sodium p-toluenesulfonate.

FIG. 8 shows the fluorescence from reticulocytes for in abnormal bloodsample, after being treated with a composition of the inventioncontaining the dye ReticRed3 and mixed for about 1 sec with no furtherincubation. The percentage of reticulocytes enumerated by this methodwas 1%. An independent reference value for reticulocyte percentageobtained for this sample by a conventional Retic-Count™ method wasapproximately 1.3%. The reagent composition in this embodiment includedIgepal, Dodecyl-β-D-maltoside and sodium p-toluenesulfonate.

FIG. 9A is the comparison of the fluorescence spectra of the dye Dye-1described in example 1B, in PBS solution with or without RNA present inthe solution.

FIG. 9B is the comparison of the fluorescence spectra of the dye Dye-1described in example 1B, in PBS solution with or without DNA present inthe solution.

FIG. 9C is the comparison of the fluorescence spectra of the dye Dye-2described in example 1B, in PBS solution with or without RNA present inthe solution.

FIG. 9D is the comparison of the fluorescence spectra of the dye Dye-2described in example 1B, in PBS solution with or without DNA present inthe solution.

FIG. 9E is the comparison of the fluorescence spectra of the Dye-3described in example 1C, in PBS solution with or without RNA present inthe solution.

FIG. 9F is the comparison of the fluorescence spectra of the Dye-3described in example 1C, in PBS solution with or without DNA present inthe solution.

FIG. 9G is the comparison of the fluorescence spectra of the Dye-4described in example 1C, in PBS solution with or without RNA present inthe solution.

FIG. 9H is the comparison of the fluorescence spectra of the Dye-4described in example 1C, in PBS solution with or without DNA present inthe solution.

FIG. 9I is the comparison of the fluorescence spectra of the Dye-5described in example 1C, in PBS solution with or without RNA present inthe solution.

FIG. 9J is the comparison of the fluorescence spectra of the Dye-5described in example 1C, in PBS solution with or without DNA present inthe solution.

FIG. 9K is the comparison of the fluorescence spectra of the Dye-6described in example 1C, in PBS solution with or without RNA present inthe solution.

FIG. 9L is the comparison of the fluorescence spectra of the Dye-6described in example 1C, in PBS solution with or without DNA present inthe solution.

FIGS. 10A, 10B, and 10C show the fluorescence from reticulocytes forthree different blood samples (one normal, two abnormal high retics)which were treated with the dye Dye-1 in an isotonic saline solution inthe presence of a rapid staining composition, and analyzedinstantaneously thereafter. In these examples the reagent compositionused to facilitate rapid staining included Igepal®, andDodecyl-β-D-maltoside.

FIGS. 11A, 11B, and 11C show the fluorescence from reticulocytes forthree different blood samples (one normal, two abnormal high retics)which were treated with the dye Dye-2 in an isotonic saline solution inthe presence of a rapid staining composition, and analyzedinstantaneously thereafter. In these examples the reagent compositionused to facilitate rapid staining included Igepal®, andDodecyl-β-D-maltoside.

FIGS. 12A, 12B, and 12C show the fluorescence histograms of red bloodcells containing different numbers of reticulocytes. For these threedifferent blood samples (one normal, two abnormal high retics), eachsample was treated with the Dye-3 in an isotonic saline solution in thepresence of a rapid staining composition, and analyzed on a BECKMANCOULTER™ XL™ flow cytometer instantaneously thereafter. In this examplethe reagent composition used to facilitate rapid staining included thedetergent Igepal, and the surfactant Dodecyl-β-D-maltoside.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides dyes for staining nucleic acids, as wellas compositions and methods to facilitate rapid transport of dyesthrough cell membranes for staining reticulocytes. Advantageously, thedyes of the invention are characterized by exhibiting significantlystronger fluorescence when bound to nucleic acids than when unbound.

In a preferred embodiment, suitable samples containing reticulocyte cellpopulations for analyses using the dyes, compositions and methods of thepresent invention are preferably selected from whole blood, or bloodsamples that have been enriched or depleted by certain additionalprocesses prior to staining. Such enrichment or depletion processes mayinclude, among others, centrifugation, ficoll assisted gravityseparation or magnetic separation.

Advantageously, the dyes, methods and compositions of the invention donot require cell fixation and are therefore particularly well suited foruse in analyses of metabolically active cells. However, selection of thecell population is not a limitation on the present invention.

As discussed above, only a few dyes, which have the ability to fluorescein the red or blue region, are suitable for reticulocyte enumeration.These dyes, however, require the use of elevated temperatures,necessitate long incubation periods, create large amounts ofnon-specific background fluorescence, and/or show little enhancement offluorescence in presence of RNA. The present invention provides noveldyes, both red and blue-excitable, which bind nucleic acids and permitdetection of reticulocytes via fluorescence at ambient temperatures.Suitably, the dyes of the invention are adapted to methods whichpreferentially permit rapid detection of reticulocytes in the absence ofsubstantial background fluorescence. An advantage of the method of theinvention is the speed at which the staining is accomplished and thefact that it can be achieved using whole blood at room temperature.

The present invention provides three groups of novel dyes that are setforth as:

Group I having the general formula of:

wherein n is 0, 1, 2, or 3; R₁ is H, alkyl, or an alkoxy group; R₂ isCH₂(CH₂)_(m)OH,

wherein m is 0, 1, 2, or 3; X is O, S, or C(CH₃)₂; R is CH₃, CH(CH₃)₂,CH₂CH₂OH, alkyl, alkylsulfonate, or hydroxyalkyl; and B− is acounteranion.

Group II having the general formula of:

wherein, n is 0, 1, 2, or 3; R1 is H, alkyl, or an alkoxy group; R2 isCH₂(CH₂)_(m)OAc, wherein m is 0, 1, 2, or 3; X is O, S, or C(CH₃)₂; R isCH₃, CH(CH₃)₂, CH₂CH₂OAc, alkyl, alkylsulfonate, or hydroxyalkyl; and B−is a counteranion.

Group III having the general formula of:

wherein n is 0, 1, or 2; R1 is H, alkyl, or an alkoxy group; R2 isCH₂(CH₂)_(m)OH, or CH₃ and wherein m is 0, 1, 2, or 3; X is O, S, orC(CH₃)₂; B⁻ is a counter anion, and R3, R4, R5, R6 are varioussubstituents as represented by the formulae for the specific compoundsDye-3 through Dye-6.

According to the invention, a counterion includes, without limitation, asingle element or a negatively charged group. In one embodiment of theinvention, the counteranion is a halide selected from among Br⁻, Cl⁻, orI⁻. In another embodiment of the invention, the counteranion is atosylate group (OTs⁻), wherein OTs⁻ is [CH₃(C₆H₄)SO₃]⁻. In still anotherembodiment of the invention, the counteranion isBF₄ ⁻. However, theinvention is not so limited. It is within the skill of one in the art toselect a suitable counteranion, from among those known ions and ionicgroups, including, without limitation, PF₆ ⁻.

This invention provides dye compounds having the general formula givenabove, which may be either red-excitable or blue-excitable. As usedherein, a red excitable dye is a dye which fluoresces when illuminatedwith light of a wavelength in the red spectral range. As used herein,this red spectral range is from about 600 nm to about 725 nm, andpreferably, 630 nm to 670 nm. A blue excitable dye is a dye whichfluoresces when contacted with light of a wavelength in the bluespectral range. As used herein, a blue excitable dye can typically havean absorption maximum in the range of 420 nm to 560 nm, and preferablyfrom about 450 nm to about 520 nm.

As stated above, the dyes of the invention show strong enhancement offluorescence when bound to nucleic acids. Generally, the dyes of theGroup I general formula have a fluorescence enhancement ratio of greaterthan about 200 (ratio of fluorescence intensity of RNA-bound dye tofluorescence intensity of free dye). In some embodiments, the dyes ofthe invention have a fluorescence enhancement ratio of greater thanabout 300. Still other dyes of the invention will have a fluorescenceenhancement ratio of greater than about 350, yet other dyes of theinvention will have a fluorescence enhancement ratio of greater thanabout 400, and still other dyes of the invention will have a have afluorescence enhancement ratio of greater than about 450. For specificexamples, see, e.g., FIG. 1B and Table 1. However, the present inventionis not limited by the ratios provided herein.

In one desirable embodiment, the invention provides a dye, termedherein, ReticRed1 having the following formula.

ReticRed1 is further described in Example 1A, compound (6). Withreference to the general formula, in ReticRed1, n is 1, R1 is H,R2=CH₂CH₂OH, R=CH₂CH₂OH, X is S, and B⁻ is Br−.

In another desirable embodiment, the invention provides a dye, termedReticRed2, having the formula:

With reference to the general formula, in ReticRed2 n is 1, R₁ is H, Ris CH(CH₃)₂, X is S, R₂ is CH₂CH₂OH, and B⁻ is Br⁻.

In yet another desirable embodiment, the invention provides a dye,termed ReticRed3, having the formula:

With reference to the general formula, in ReticRed3 n is 1, R₁ is H, Ris CH₃, R₂ is CH₂CH₂OH, X is S, and B⁻ is Br⁻.

In still another desirable embodiment, the invention provides a dye,termed ReticRed4, having the formula:

With reference to the general formula, in ReticRed4, n is 1, R₁ is CH₃,R is CH₃, R₂ is CH₂CH₂OH, X is S, and B⁻ is Br⁻.

In a further embodiment, the invention provides a dye, termed ReticRed5,having the formula:

With reference to the general formula, in ReticRed5, n is 1, R₁ is CH₃,R is CH(CH₃)₂, R₂ is CH₂CH₂OH, X is S, and B⁻ is Br⁻.

In another desirable embodiment, the invention provides a dye, termedReticRed6, having the formula:

With reference to the general formula, in ReticRed6, n is 1, R₁ is CH₃,R is CH₂CH₂OH, R₂ is CH₂CH₂OH, X is S, and B⁻ is Br⁻.

In yet another desirable embodiment, the invention provides ablue-excitable dye, termed ReticBlue1, where with reference to thegeneral formula, n is 0. One particularly desirable example of apreferred blue excitable dye is ReticBlue1, which is represented by thegeneral formula, wherein, n=0, R1=H, m=1, R2=CH₂CH₂OH, R=CH₂CH₂OH, X=S,B⁻=Br⁻

In one desirable embodiment, the invention provides a dye, termedherein, Dye-1 having the following formula:

With reference to the Group-II General Formula, the Dye-1 has n−1; R₁=H;R=CH₃; R₂=CH₂CH₂OAc; X=S; and B⁻ is Br^(−.)

In another desirable embodiment, the invention provides a dye, termedDye-2, having the formula:

Dye-2 is further described in Example 1B. With reference to the Group-IIGeneral Formula, the Dye-2 has n=1, R1=H, R2=R=CH₂CH₂OAc, X=S, and B⁻ isBr⁻.

In another desirable embodiment, the invention provides a dye, termedherein, Dye-3 having the following formula:

Dye-3 is further described in Example 1C. With reference to theGroup-III General Formula, the Dye-3 has n=1, X is S, R1=H, R2=CH₂CH₂OH,R3═R5=OCH₃, R4═R6=H, and B⁻ is Br⁻.

In another desirable embodiment, the invention provides a dye, termedDye-4, having the formula:

Dye-4 is further described in Example 1C. With reference to theGroup-III General Formula, the Dye-4 has n=1; X=S; R1=H; R2=CH₂CH₂OH;R3=R5=R6=H; R4=CO.CH₃; and B⁻ is Br⁻.

In another desirable embodiment, the invention provides a dye, termedDye-5, having the formula:

Dye-5 is further described in Example 1C. With reference to theGroup-III General Formula, the Dye-5 has n=1; X=S; R1=H; R2=CH₂CH₂OH;R3=R4=R5=R6=H; and B⁻ is Br⁻.

In yet another desirable embodiment, the invention provides a dye,termed Dye-6, having the formula:

Dye-6 is further described in Example 1C. With reference to theGroup-III General Formula, the Dye-6 has n=1; X=S; R1=H; R2=CH₃;R3=R4=R5=H; R6=B(OH)₂; and B⁻ is I⁻.

The dyes of the invention are useful for a variety of purposes whichwill be readily apparent to those of skill in the art. Such usesinclude, for example, use of dyes as markers or tags for detecting thepresence of a molecule or compound to which they are bound. Such markersmay be used for monitoring the efficacy of a therapeutic compound invivo or for diagnostic uses, or the like. However, the dyes of theinvention are particularly well suited for staining of nucleic acids.For example, these dyes are particularly suitable for staining of RNA inreticulocytes. In another exemplary application, these dyes are suitablefor staining DNA in nucleated red blood cells. Typically, when used instaining of nucleic acids, the dyes are formulated into reagentsolutions.

I. Staining Reagents

The invention provides a variety of compositions, described herein,which are useful as staining reagents in a variety of applications,including a variety of assay formats. Examples of such uses will bereadily apparent to those of skill in the art, upon a review of thecompositions described herein.

A. Dye Compositions

In order to utilize the novel dyes of the invention for staining ofnucleic acids, the dyes are dissolved in an appropriate solvent to formsolutions. As defined herein, a solvent is meant to describe any liquidthat can dissolve a solid, liquid, or gas. A preferred embodiment of theinvention utilizes water-based or miscible liquids, such asdimethylsulfoxide (DMSO), methanol, ethanol, and mixtures thereof.Selection of a suitable solvent is however not a limitation on thepresent invention. For storage, typically a relatively highconcentration of the dye is dissolved in a suitable solvent to obtain astock solution. In a preferred embodiment, to obtain a stock solution ofthe dye, the dye is dissolved in DMSO at a dye concentration rangingfrom 0.5 mM to 10 mM, and most preferably, 1 to 5 mM.

For further application of the dye composition in the method of theinvention, the stock solution may be diluted in other reagents orbuffers, including, for example, water, saline, phosphate bufferedsaline (PBS), or isotonic saline. The final dye concentration in suchdye compositions may be varied depending on the application. In oneembodiment, the concentration of the dye in the final dye compositionadded to a sample of whole blood is in the range of about 0.1-50 μM,preferably from about 0.5 to 25 μM, and more preferably 2 to 10 μM.However, selection of an appropriate concentration or diluting solventis not a limitation on the present invention.

The dye composition of the invention can be utilized for a variety ofpurposes. A preferred embodiment of this invention is the use of the dyecomposition for staining, detection, and analysis of nucleic acids.Another preferred embodiment of this invention is the use of the dyecomposition for staining, detection, and analysis of reticulocytes inwhole blood. Yet another preferred embodiment of this invention is theuse of the dye for staining, detection and analysis of nucleated redblood cells in whole blood. One of skill in the art will readilyunderstand that use of the dyes and reagents of the present invention isnot so limited. In one particularly desirable embodiment, for example,where the dye contacts intact cells, it can be desirable to formulateone or more of the dyes into a mixture.

B. Compositions for Rapid Staining

In another aspect, a dye composition of the invention may be utilized tofacilitate rapid transport of the dyes through the cell membrane,thereby permitting the dye to stain reticulocytes in about 1 minute orless. Thus, the method of the invention can permit staining in about 0to about 60 seconds, preferably from about 0 seconds to about 45seconds, more preferably from about 0 seconds to about 30 seconds, mostpreferably from about 0 seconds to about 20 seconds.

Generally, such rapid staining requires that a sample be contacted witha dye composition of the invention in the presence of at least onesurfactant and optionally, a sulfonic acid or a salt thereof.

When used for rapid staining, additional components may be added to thecomposition of the invention. For example, buffers may be employed insituations where maintaining the pH of the dye composition is necessary.Preferably, the pH of the dye reagent is maintained in the range ofabout 6 to about 9. More preferably, a pH in the range of about 7 toabout 7.5 is obtained. The buffer may be selected from a variety ofbuffers known to those of skill in the art to be used in thecompositions of the invention and include, without limitation, phosphatebuffered saline (PBS) or isotonic saline, such as ISOTON®II diluent,U.S. Pat. No. 3,962,125, [Beckman Coulter, Inc., Miami, Fla.], or thelike. Additionally, such buffers may also be used to adjust theconcentration of one or more of the components of the composition ofthis invention. Preservatives may also be added to the compositions ofthe invention, and may be selected from, but is not limited to,5-Chloro-2-methyl-4-isothiazolin-3-one, and2-methyl-4-isothiazolin-3-one [such preservatives may be purchasedcommercially, e.g., as ProClin 300 or ProClin 150].

At least one surfactant is used in the rapid staining composition. Inone exemplary embodiment, the surfactants include a detergent and asecond surfactant which functions as a sphering agent for a cell. Inanother exemplary embodiment, the surfactants may be designed such thata single surfactant functions as both a detergent and a sphering agent.In yet another exemplary embodiment, the surfactants may be designedsuch that a single surfactant provides the necessary surfactantfunction, but no sphering reagent function is required.

Surfactants to be used in the composition of the invention may beselected from among the anionic surfactant ammonium perfluoralkylcarboxylate [commercially available as Fluorad® FC-143 (3M Company,Minneapolis, Minn.)], sodium lauroyl myristoyl lactylate [commerciallyavailable as Pationic® 138C (R.I.T.A. Corp, Woodstock, Ill.)], or fromthe non-ionic surfactants Dodecyl-β-D-maltoside,N,N-bis[3-D-gluconamidopropyl] cholamide,polyoxypropylene-polyoxyethylene block copolymer,N-Tetradecyl-β-D-maltoside, Daconyl-N-methyl-glucamide,n-Dodecyl-β-D-glucopyranoside, n-Decyl-β-D-glucopyranoside, polyethyleneglycol ester of stearic acid, ethoxylated cocomonoglyceride,octyphenoxypoly (ethyleneoxy) ethanol, ethoxylated octylphenol, andlinear alcohol, or, from among the cationic surfactants, cocohydroxyethyl imidazoline, lauryltrimethylammonium chloride,decyltrimethylammonium bromide, octyltrimethylammonium bromide, or fromamong the zwitterionic surfactants lauramidopropyl betaine,N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,cocoamidopropylbetaine, cocoamidosulfobetaine,N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate.

As discussed above, the surfactant selected may function as a spheringagent for red cells when the concentration and osmolarity isappropriately adjusted. A sphering agent may be readily selected by oneof skill in the art. A preferred sphering reagent is based on thenon-ionic surfactant Dodecyl-β-D-maltoside, which suitably is insolution with a buffer such as phosphate buffered saline. To effectivelyisovolumetrically sphere the reticulocytes and red blood cells within ablood sample, the concentration of the sphering reagent in thecomposition is most preferably from about 3 μg/ml to about 50 μg/ml witha mOsm in the range of about 200 to about 400 mOSm, and preferably fromabout 250 mOsm to about 350 mOsm. However, one of skill in the art mayreadily adjust this concentration and osmolarity as needed or desired toisovolumetrically sphere the cells, taking into consideration thesurfactant selected. As discussed above, optionally, the selection ofthe sphering reagent may eliminate the need for the detergent.

In one embodiment, a detergent is included in the dye composition of theinvention. Detergents to be used are selected from among non-ionicdetergents. Desirably, these detergents are used at a concentrationbetween about 0 to about 1% (w/v). In one preferred embodiment of theinvention, the detergents are used at concentrations of between about0.001% to about 0.5%, and in a more preferred embodiment are in therange from about 0.005% to 0.1%. One currently preferred detergent isoctylphenoxypoly(ethyleneoxy)ethanol [commercially available as Igepal®CA-630 (Sigma N-6507) or Nonidet P-40 (Sigma)]. Examples of othersuitable detergents include, ethyoxylated octylphenol [commerciallyavailable as Triton X-100 (Sigma T9284)], and linear alcohol alkoxylates[commercially available as Plurafac® A-38 (BASF Corp) or Plurafac® A-39(BASF Corp)]. Typically, these detergents are mixed with a sample (orvice versa), e.g., 1-2 μl of whole blood or the like, or separatelyformulated into a composition of the invention.

In one desirable embodiment, a sulfonic acid, or a salt thereof, isincluded in the composition of the invention. The sulfonic acid or saltthereof acts as a further facilitator of transporting the dyecomposition through the cell membrane and is used at a concentrationranging from about 0.01 to about 250 μM, most preferably from 0.01 toabout 50 μM. One embodiment of this invention utilizes p-toluenesulfonicacid. In other embodiments of this invention, a p-toluenesulfonate saltcan be substituted for p-toluenesulfonic acid. Examples of such saltsinclude sodium, potassium, silver, zinc, and barium p-toluenesulfonatesalts. Some examples of such salts are commercially available, e.g.,from Sigma-Aldrich. Other suitable sulfonates may be readily selectedfor use in the invention.

The dye compositions of the invention can contain a single dye,optionally in the presence of other dye. In one embodiment of theinvention, a composition of the invention can contain one or more of thedye compounds of this invention, or any combination thereof.

In yet another embodiment of the invention, the composition of theinvention can contain other dyes known to those of skill in the art incombination with one or more of the novel dyes of the invention. Suchdyes are readily selected from among dyes which have been publishedand/or which are commercially available. See, for example, BeckmanCoulter, Inc. (Fullerton, Calif.) catalog. There are a variety of usesfor the dyes and compositions of the invention, which will be readilyapparent to one of skill in the art.

In yet another embodiment of the invention, the composition of theinvention can contain other dyes and reagents known in the art asblocking agents for specific nucleic acid or proteins within cells, incombination with one or more of the novel dyes of the invention. Suchblocking agents are readily selected from among dyes and reagents whichhave been published and/or which are commercially available. See, forexample, Beckman Coulter, Inc. (Fullerton, Calif.) catalog or MolecularProbes (Eugene, Oreg.) catalog. There are a variety of uses for the dyesand compositions of the invention, which will be readily apparent to oneof skill in the art.

II. Methods of Staining Nucleic Acids Using the Dyes of the Invention

The dye composition of the invention is useful in staining a variety ofnucleic acids, including DNA and RNA.

In samples containing free nucleic acids, for example, nucleic acids notsurrounded by an intact cell membrane of a cellular or non-cellularsource, the dye composition of the invention can stain the nucleicacids. In one embodiment of the invention, a method is provided forcontacting a sample containing nucleic acids with the dye compositionsof the invention. Preferably, the method involves contacting a samplecontaining nucleic acids with a dye composition of the invention,optionally in the presence of other dyes and reagents known to those ofskill in the art. In a preferred embodiment of the invention, the methodinvolves contacting a sample containing nucleic acids with a dyecomposition of the invention, which contains about 2 to about 10 μm redexcitable ReticRed1 dye and an isotonic aqueous buffer. In anotherpreferred embodiment of the invention, the method involves contacting asample containing nucleic acids with a dye composition of the inventionwhich contains about 1 to about 10 μM of red excitable Dye-1, Dye-2,Dye-3 or combinations thereof and an isotonic aqueous buffer.

The dye composition of the invention are particularly well suited foruse in the detection and enumeration of reticulocytes, which containRNA. The method of the invention therefore involves contacting areticulocyte, containing nucleic acid, with one or more dye compositionsof the invention.

The dye composition of the invention, preferably Groups II and III, arealso well suited for use in the detection and enumeration of nucleatedred blood cells, which contain DNA.

In one embodiment, the method involves contacting the blood cells with adye composition of the invention in the presence of at least onesurfactant, optionally together with a preservative and sulfonicreagent. As described above, the surfactant(s) can include a detergentand/or a sphering agent. According to the method of the invention, thecells can be contacted with a composition containing these components.Alternatively, one or more of these components can be deliveredseparately, for example by adding the component(s) directly to thesample. The mixture is then incubated for a suitable period of time. Aspreviously discussed, the incubation time will be less than 1 minute.However, if desired for purposes of convenience, the incubation periodmay be either extended or shortened. For example, by adjusting theconcentration of the dye, surfactant or detergent, the incubation timecan be increased to greater than 1 minute. Desirably, this mixing andincubation may be performed at temperatures between approximately 20° C.to 40° C. However, in a preferred embodiment of this invention,temperatures ranging from 22° C. to 28° C., may be utilized.

In a currently preferred embodiment, the method of the inventionrequires incubation of the dye for a period ranging from above 0 secondsto about 1 minute to stain reticulocytes, and the staining can beaccomplished at room temperatures. In another preferred embodiment,incubation time is eliminated because the blood sample is mixed with thedye composition and thereafter immediately analyzed by the instrument.See Example 5, FIG. 7 and FIG. 8 and Example 7, FIGS. 10A-2B, 11A-11C,12A-12C. Prior art staining techniques using red-excitable dyes forreticulocytes in this time frame require incubation at elevatedtemperatures and/or additional use of toxic ionophoric compounds.

Reticulocytes stained with the dye compositions, according to the methodof the invention, are preferably enumerated in an automatic flowcytometer. However, these cells may also be counted by a manualprocedure or automated microscopy.

Thus, the method of the invention facilitates transport of the nucleicacid-specific dyes of this invention across the cell membrane in thetime frame desirable for automated flow cytometry. Automatic flowcytometers are well known in the art, and the present invention is notlimited to the use of any particular flow cytometer. A preferredembodiment of the invention entails the detection and enumeration ofreticulocytes using the XL™ flow cytometer [Beckman Coulter Inc., Miami,Fla.]. Different analytical techniques can be employed in theenumeration of reticulocytes by flow cytometric measurements including,but not limited to light scatter, fluorescence, optical absorption,axial light loss, DC electrical impedance, and radio frequency (RF)conductivity. In a preferred embodiment of the invention, in using suchflow cytometers, light scatter gates are used to isolate red cells, andfluorescent gates are then used to delineate reticulocytes from maturered cells and enumerate the reticulocytes. In another embodiment of theinvention, using flow cytometers, DC and light scatter gates are used toisolate red cells, and fluorescent gates are then used to delineatereticulocytes from mature red cells and enumerate the reticulocytes. Inyet another embodiment of the invention, using flow cytometers, DC andfluorescent measurement alone are used to delineate reticulocytes frommature red cells and enumerate the reticulocytes.

Another notable advantage of the preferred embodiment of the inventionis that when the composition includes a red-excitable dye of theinvention, such as ReticRed1, ReticRed3, Dye-1, Dye-2 or Dye-3, itpermits the use of a less expensive excitation source than argon lasersused for other presently available reticulocyte dyes, such as CPO, AO[Seligman, Am. J. Hematology, 14:57 91983)], TO [Lee et. al., Cytometry,7:508 (1986)], Auramine-O and Pyronin-Y. For example, when thered-excitable dye composition of the invention is used, the presentinvention advantageously permits the use of a red laser as theexcitation source. Specifically, a red diode laser, a high power lightemitting diode (LED), or a red helium-neon laser can be used to conductreticulocyte counting using a red-excitable dye of this invention.

A variety of lasers are known to those of skill in the art that may beutilized as the excitation sources for the flow cytometers. The lasersmay be selected from, but are not limited to, argon, helium-neon, diode,and diode pumped solid-state lasers, depending upon the excitationwavelength of the dye of the invention selected for detection ofreticulocytes. Selection of suitable light sources, and appropriateexcitation wavelengths, are not a limitation on the present invention.

Suitable excitation wavelengths may be readily determined by one ofskill in the art. Examples of suitable excitation wavelengths in the redspectral range include those in the range of 600 nm to 725 nm, andpreferably 630 nm to 670 nm. Other suitable excitation sources andwavelengths may be readily selected by one of skill in the art, takinginto consideration the dye selected for use in the method andcompositions of the invention.

An additional advantage comes from the fact that red-excitable dyecompounds of this invention such as ReticRed1, ReticRed2, ReticRed3,ReticRed4, ReticRed5, ReticRed6, Dye-1, Dye-2 or Dye-3 etc., areessentially non-fluorescent or very weakly fluorescent in aqueoussolutions in the absence of nucleic acids (unbound state). As a result,problems associated with background fluorescence are minimal, andreticulocytes can be detected with high sensitivity using this dye.

Thus, the present invention permits reticulocytes to be rapidly stainedwith the dyes of the invention for subsequent flow analysis. The methodof the invention differs from both the prior art Pyronin-Y [Tanke et al,cited above] staining procedure requiring fixation of the cells and theprior art fluorescence staining procedures involving membrane permeabledyes such as CPO (reticONE™) or Thiazole Orange (Retic-Count™) that doesnot require cell-fixation, but requires long incubation with the bloodto accomplish staining.

III. Examples

The following examples are provided to illustrate the invention and donot limit the scope thereof. One skilled in the art will appreciate thatalthough specific reagents and conditions are outlined in the followingexamples, modifications can be made which are meant to be encompassed bythe spirit and scope of the invention.

Example 1A—Materials and Methods for Synthesis of Group I Compounds

A. General Dye Synthesis for Group I

B. Specific Dye Preparation

Preparation of 1-(2-Hydroxy)ethyllepidinium Bromide (2). A solution oflepidine (11.5 g) and bromoethanol (100 g) were stirred and heated in anoil bath at 110° C. for 48 hours. Cooling the reaction mixture to roomtemperature, adding ethyl acetate (100 ml) and stirring for 5 minresulted in a solid that was collected, washed with ethyl acetate (2×20ml) and dried. The solid was then dissolved in methanol (80 ml) andprecipitated with ethyl acetate (600 ml). The solid was collected viafiltration, washed with ethyl acetate (2×100 ml) and dried in an ovenunder high vacuum at 50° C. overnight to obtain 11.47 g (53% yield) ofproduct (2 c).

Preparation of 3-(2-Hydroxy)ethyl-2-methylbenzothiazolium Bromide (4 a).A solution of 2-methylbenzothiazole (5.87 g) and 2-bromoethanol (49.2 g)was stirred and heated in an oil bath at 110° C. for 18 hours. Aftercooling the reaction mixture to room temperature, adding ethyl acetate(100 ml), and decanting, the resultant solid was then dissolved inmethanol (50 ml) and precipitated with ethyl acetate (300 ml). The solidwas again recrystallized from a mixture of methanol and ethyl acetate,washed with ethyl acetate (2×25 ml) and dried in an oven under highvacuum at 50° C. overnight to obtain 4.21 g (39%) of 4 a.

Preparation of 3-(2-Hydroxy)ethyl-2,6-dimethylbenzothiazolium Bromide (4b). A solution of 2,6-dimethylbenzothiazole (13.0 g) and 2-bromoethanol(100 g) was stirred and heated in an oil bath to 120° C. for 96 hours.After cooling the reaction mixture to room temperature, adding ethylacetate (150 ml) and stirring for 1 hour, the resultant solid wasfiltered and collected. The solid was then dissolved in methanol (150ml) and charcoal (2 g) was added. The charcoal was removed by passingthrough a pad of Celite®. The solution was then concentrated to a smallvolume and triturated with acetone (200 ml). The solid was collected,washed with acetone (2×30 ml), ethyl acetate (2×30 ml) and dried in anoven under high vacuum at 50 C overnight to obtain 13.95 g (61%) of 4 b.

Preparation of 3-(2-Hydroxy)ethyl-6-methoxy-2-methylbenzothiazoliumBromide (4 c). A solution of 6-methoxy-2-methylbenzothiazole (1.82 g)and 2-bromoethanol (8.8 g) was stirred and heated in an oil bath at 120°C. for 74 hours. After cooling the reaction mixture to room temperature,adding ethyl acetate (25 ml) and stirring for 1 hour, the solid wasfiltered and collected. The solid was then dissolved in methanol (50 ml)and charcoal (0.5 g) was added. The charcoal was removed by passingthrough a pad of Celite®. The solution was then concentrated to a smallvolume and triturated with ethyl acetate (100 ml). The solid wascollected, washed with ethyl acetate (2×30 ml) and dried in an ovenunder high vacuum at 50° C. overnight to obtain 0.9 g (30%) of 4 c.

Preparation of 3-(2-Hydroxy)ethyl-2-(2-N-phenyl)ethenylbenzothiazoliumBromide (5 a). To a solution of3-(2-hydroxy)ethyl-2-methylbenzothiazolium bromide (4 a, 1.19 g) in amixed solvent of methanol/ethanol (3:1, 80 ml) was added an excess ofethyl N-phenylformimidate (3.0 g) and the solution was stirred at roomtemperature for 36 h. The solvent was removed under reduced pressure togive a dry solid. The solid was subjected to column chromatography(silica gel, methylene chloride/methanol) purification. The fractionscontaining the product were combined and concentrated to dryness underreduced pressure. The solid was dissolved in methanol (10 ml) andprecipitated with ethyl ether (200 ml). After drying at 50° C. underhigh vacuum overnight, 1.04 g (63%) of 5 a was obtained as a yellowsolid.

Preparation of3-(2-Hydroxy)ethyl-6-methyl-2-(2-N-phenyl)ethenylbenzothiazolium Bromide(5 b). To a solution of 3-(2-hydroxy)ethyl-2,6-dimethylbenzo-thiazoliumbromide (4 b, 2.76 g) in methanol (80 ml) was added an excess of ethylN-phenylformimidate (3.6 g) and the solution stirred at room temperaturefor 36 hours. Ethyl acetate (160 ml) was then added and stirredovernight. The solid was collected, washed with ethyl acetate (2×30 ml)and dried. Drying the solid further in an oven at 50° C. under highvacuum overnight afforded 2.32 g (62%) of 5 b as a yellow solid.

Preparation of3-(2-Hydroxy)ethyl-6-methoxy-2-(2-N-phenyl)ethenylbenzothiazoliumBromide (5 c). To a solution of3-(2-hydroxy)ethyl-6-methoxy-2-methylbenzothiazolium bromide (4 c, 0.79g) in methanol (30 ml) was added an excess of ethyl N-phenylformimidate(1.5 g) and the solution was stirred at room temperature for 16 h. Thesolvent was removed under reduced pressure to yield a dry solid. Thesolid was subjected to column chromatography (silica gel), and elutedwith a methanol/methylene chloride gradient (from 0 to 15% methanol).The fractions containing the product were combined and concentrated todryness under reduced pressure. The solid was dissolved in methanol (5ml) and precipitated with ethyl ether (150 ml). After drying in an ovenat 50° C. under high vacuum overnight, 0.35 g (33%) of 5 c as a yellowsolid was obtained.

Preparation of Dye Compound 6 (Retic Red1). To a solution of3-(2-hydroxy)ethyl-2-(2-N-phenyl)ethenylbenzothiazolium bromide (5 a,85.8 mg) and 1-(2-hydroxy)ethyllepidinium bromide (2, 58.4 mg) inmethylene chloride (20 ml) was added triethylamine (91 μL) and aceticanhydride (62 μL). After stirring at room temperature for 2 hours thesolvent was removed under reduced pressure to give a blue solid. Thesolid was partially redissolved in methanol (15 ml) and ethyl acetate(300 ml) was added. The resultant solid was collected via filtration,washed with ethyl acetate (2×30 ml) and dried. Further drying the solidin an oven at 50° C. under high vacuum overnight afforded 67.8 mg (66%)of 8 as a blue solid. Absorption spectrum maximum: 630 nm (in methanol),640 nm (in DMSO).

Preparation of Dye Compound 7 (ReticRed6). To a solution of3-(2-hydroxy)ethyl-6-methyl-2-(2-N-phenyl)ethenylbenzothiazolium bromide(5 b, 85.6 mg), 1-(2-hydroxy)ethyllepidinium bromide (2, 58.7 mg) inmethylene chloride (10 ml), and methanol (1.0 ml) was addedtriethylamine (91 μL) and acetic anhydride (61 μL). After stirring atroom temperature for 2 hours the solvent was removed under reducedpressure to give a blue solid. The solid was partially redissolved inmethanol (10 ml) and ethyl acetate (200 ml) was added. The resultantsolid was collected via filtration, washed with ethyl acetate (2×30 ml)and dried. Further drying the solid in an oven at 50° C. under highvacuum overnight afforded 63.7 mg (60%) of 11 as a blue solid.Absorption spectrum maximum: 636 nm (in methanol), 645 nm (in DMSO).

Example 1B: Materials and Methods for Synthesis of Group-II Compounds

(i) Preparation of 1-Methyllepidinium Iodide (2 a). A solution oflepidine (10.56 g) and methyliodide (105 g) was stirred and heated toreflux in an oil bath at 50° C. for 19 hours. Cooled the reactionmixture to room temperature, added acetone (200 mL) and stirred for 2hours. The resultant solid was collected, washed with ethyl acetate(2×20 mL) and then triturated with ethyl acetate (200 mL). The solid wascollected via filtration, washed with ethyl acetate (2×25 mL) and driedin an oven under high vacuum at 50° C. overnight. There was obtained20.74 g (98.6% yield) of product.

(ii) Preparation of 1-(2-Hydroxyethyl)lepidinium Bromide (2 b). Asolution of lepidine (11.5 g) and bromoethanol (100 g) was stirred in anoil bath at 110° C. for 48 hours. Cooled the reaction mixture to roomtemperature, added ethyl acetate (100 mL) and stirred for 5 min. Theresultant solid was collected, washed with ethyl acetate (2×20 mL) anddried. The solid was then dissolved in methanol (80 mL) and precipitatedwith ethyl acetate (600 mL). The solid was collected via filtration,washed with ethyl acetate (2×100 mL) and dried in an oven under highvacuum at 50° C. overnight. There was obtained 11.47 g (53% yield) ofproduct.

(iii) Preparation of 3-(2-Hydroxyethyl)-2-methylbenzothiazolium Bromide(4). A solution of 5.87 g of 2-methylbenzothiazole (3) and bromoethanol(49.2 g) was stirred in an oil bath at 110° C. for 18 hours. Added ethylacetate (100 mL) to the reaction mixture and decanted. The residualsolid was then dissolved in methanol (50 mL) and precipitated with ethylacetate (300 mL). The solid was again recrystallized from methanol-ethylacetate (15 mL/150 mL), washed with ethyl acetate (2×25 mL) and dried inan oven under high vacuum at 50° C. overnight. The yield was 4.21 g(39%). TLC (silica gel, 4:1 methylene chloride:methanol) R_(f)=0.34.

(iv) Preparation of 3-(2-Hydroxyethyl)-2-(2-N-phenyl)ethenylbenzothiazolium Bromide (5). To a solution of3-(2-hydroxyethyl)-2-methylbenzothiazolium bromide (4, 1.19 g) in amixed solvent of methanol/ethanol (3:1, 80 mL) was added excess of ethylN-phenylformidiate (3.0 g) and stirred at room temperature for 36 hours.Evaporated off the solvent under reduced pressure to dryness. The solidwas subjected to column chromatography (silica gel, methylenechloride/methanol) purification. The fractions contained the productwere combined and concentrated to dryness under reduced pressure. Thesolid was dissolved in methanol (10 mL) and precipitated with ethylether (200 mL). After drying in an oven at 50° C. under high vacuumovernight there was obtained 1.04 g (63%) of a yellow solid. TLC (silicagel, 9:1 methylene chloride:methanol) R_(f)=0.27.

(v) Preparation of dye compound corresponding to the configurationdescribed as 6 in the above figures, having R=CH₃, R1=H. A solution of3-(2-hydroxyethyl)-2-(2-N-phenyl)ethenylbenzothiazolium bromide (5,203.8 mg) and 1-methyllepidinium iodide (2 a, 154.0 mg) in pyridine (10mL) was heated in an oil bath at 90° C. for 2 hours under anhydrouscondition. A solution of sodium tetrafluoroborate (59.3 mg) in DMF (0.5mL) was added and the heating continued for another 20 min at 90° C.After cooling to room temperature the reaction solution was poured intoethyl acetate (100 mL) and the resultant solid was collected viafiltration. Re-dissolved the solid in methanol (100 mL) and added ethylacetate (300 mL) and stirred overnight. The resultant solid wascollected via filtration, washed with ethyl acetate (2×20 mL) and dried.Further drying the solid in an oven at 50° C. under high vacuumovernight afforded 165.4 mg (68%) of a blue solid. TLC (silica gel, 4:1methylene chloride:methanol) R_(f)=0.65. Maximum Absorption: 629 nm (inmethanol).

(vi) Preparation of the dye compound corresponding to the configurationdescribed as 7, having R=CH₂CH₂(OH), R1=H. To a solution of3-(2-hydroxyethyl)-2-(2-N-phenyl)ethenylbenzothiazolium bromide (5, 85.8mg) and 1-(2-hydroxyethyl)lepidinium bromide (2 b, 58.4 mg) in methylenechloride (20 mL) was added triethylamine (91 μL) and acetic anhydride(62 μL). After stirring at room temperature for 2 hours the solvent wasevaporated off under reduced pressure to give a blue solid. Partiallyredissolved the solid in methanol (15 mL) and added ethyl acetate (300mL). The resultant solid was collected via filtration, washed with ethylacetate (2×30 mL) and dried. Further drying the solid in an oven at 50°C. under high vacuum overnight afforded 67.8 mg (66%) of a blue solid.TLC (silica gel, 4:1 methylene chloride:methanol) R_(f)=0.18. MaximumAbsorption: 631 nm (in methanol).

(vii) Preparation of the acetylated dye compound corresponding to themolecular configuration described as 8. A suspension of dye compounddescribed in (v) above (50 mg) in acetic anhydride (4 mL) and pyridine(1 mL) was heated at 50° C. with stirring in an oil bath under anhydrouscondition for 10 hours. After cooling to room temperature the reactionsolution was poured into ethyl acetate (100 mL) and stirred for 3 min.The resultant fine crystals were collected via filtration, washed withethyl acetate (2×20 mL) and dried in an oven at 50° C. under high vacuumovernight. There was obtained 56.5 mg (quantitative yield) of a bluecrystalline solid product. TLC (silica gel, 4:1 methylenechloride:methanol) R_(f)=0.79 Maximum Absorption: 625 nm (in methanol).This compound is referenced hereinafter as Dye-1.

(viii) Preparation of the acetylated dye compound corresponding to themolecular configuration described as 9. A suspension of dye compounddescribed in (vi) above, (377.5 mg) in acetic anhydride (8 mL) andpyridine (2 mL) was heated at 50° C. with stirring in an oil bath underanhydrous condition for 10 hours. After cooling to room temperature thereaction solution was poured into ethyl acetate (100 mL) and stirred for3 min. The resultant solid was collected via filtration, washed withethyl acetate (2×20 mL), ethyl ether (2×20 mL) and dried in an oven at50° C. under high vacuum overnight. There was obtained 411.9 mg (92.6%yield) of a blue solid product. TLC (silica gel, 4:1 methylenechloride:methanol) R_(f)=0.96. Maximum Absorption: 631 nm (in methanol).This compound is referenced hereinafter as Dye-2.

Example 1C—Materials and Methods for Synthesis of Group III Compounds

Preparation of 2-Bromomethylphenylboronic Acid (5). A suspension of2-methylphenylboronic acid (10.65 g), N-bromosuccinimide (NBS, 16.86 g)and 2,2′-azobisisobutyronitrile (AIBN, 1.708 g) in carbon tetrachloride(1000 mL) was heated in an oil bath at 82° C. to reflux for 2 hoursunder anhydrous condition. Cooled the reaction mixture to roomtemperature and filtered off the solid. Extracted the filtrate withwater (2×200 mL). The organic layer was separated, dried over MgSO₄ andconcentrated in vacuo to dryness. Redissolved the solid in methanol (100mL) and added ethyl acetate (1000 mL) to it. The resultant precipitatewas collected, washed with ethyl acetate (2×50 mL) and dried in the air.The filtrate was concentrated to a small volume and a second crop ofsolid was also collected. The combined solid was dried in an oven underhigh vacuum at 50° C. overnight. There was obtained 6.48 g (39%) ofproduct.

Preparation of 1-Benzyllepidinium Bromide (6). A solution of lepidine(37.82 g) and benzyl bromide (6.471 g) in acetonitrile (100 mL) werestirred and heated to reflux in an oil bath at 93° C. for 20 hours.Cooled the reaction mixture to room temperature, added ethyl acetate (50mL) and stirred for 30 min. The resultant solid was collected, washedwith ethyl acetate (4×20 mL) and dried in the air. The filtrate wasconcentrated to a small volume and a second crop of solid was alsocollected. The combined solid was dried in an oven under high vacuum at50° C. overnight. There was obtained 11.52 g (96.9%) of product.

Preparation of 1-(3′-5′-Dimethoxybenzyl)lepidinium Bromide (7). Asolution of lepidine (3.36 g) and 3,5-dimethoxybenzyl bromide (5.41 g)in acetonitrile (100 mL) were stirred and heated to reflux in an oilbath at 90° C. for 30 hours. Cooled the reaction mixture to roomtemperature, added ethyl acetate (50 mL) and stirred for 30 min. Theresultant solid was collected, washed with ethyl acetate (4×20 mL) anddried in the air. Redissolved the solid in methanol (200 mL) and treatedwith charcoal (5 g) and heated to reflux for 15 min to remove the darkcolor impurities. After filtering through a pad of Celite® the filtratewas concentrated to a small volume (˜20 mL) and ethyl acetate (300 mL)was added. The resultant precipitate was collected, washed with ethylacetate (2×50 mL), ethyl ether (2×20 mL) and dried in the air. Furtherdrying in an oven under high vacuum at 50° C. overnight provided 6.65 g(76%) of product.

Preparation of 1-(4′-Carboxymethylbenzyl)lepidinium Bromide (8). Asolution of lepidine (3.32 g) and methyl 4-(bromomethyl)benzoate (5.31g) in acetonitrile (100 mL) were stirred and heated to reflux in an oilbath at 90° C. for 16 hours. After cooling to room temperature thesolvent was evaporated off under reduced pressure to dryness. Theresultant solid was washed with ethyl acetate (3×100 mL). Redissolvedthe solid in methanol (100 mL) and treated with charcoal (5 g) andheated to reflux for 15 min to remove the dark color impurities. Afterfiltering through a pad of Celite® the filtrate was concentrated todryness. The solid was then triturated with acetone (100 mL). Theresultant solid was collected, washed with ethyl acetate (2×50 mL) anddried in the air. The filtrate was concentrated to a small volume and asecond crop of solid was also collected. The combined solid was dried inan oven under high vacuum at 50° C. overnight. There was obtained 7.26 g(84%) of product.

Preparation of 1-(2′-Boronic acid)benzyllepidinium Bromide (9). Asolution of lepidine (306 mg) and 2-bromomethylphenylboronic acid (417mg) in acetonitrile (12 mL) were stirred and heated to reflux in an oilbath at 90° C. for 17 hours. After cooling to room temperature thesolvent was evaporated off under reduced pressure to dryness. Washed thesolid with ethyl acetate (3×20 mL) and the solvent was decanted.Redissolved the solid in methanol (3 mL) and again concentrated todryness. Washed the solid with ethyl acetate (3×20 mL). The solid wasthen dried in an oven under high vacuum at 50° C. overnight. There wasobtained 650 mg (93.5%) of product.

Preparation of 2,3-Dimethylbenzothiazolium Iodide (11 a). A solution of2-methylbenzothiazole (5.00 g) and iodomethane (50 g) were stirred in anoil bath at 45° C. for 44 hours. After cooling to room temperature thesolid was collected through filtration and the solid was washed withethyl acetate (2×100 mL) and dried in an oven under high vacuum at 50°C. overnight. There was obtained 8.02 g (82.3% yield) of product.

Preparation of 3-(2-Hydroxy)ethyl-2-methylbenzothiazolium Bromide (11b). A solution of 2-methylbenzothiazole (5.87 g) and bromoethanol (49.2g) were stirred in an oil bath at 110° C. for 18 hours. Added ethylacetate (100 mL) to the reaction mixture and decanted. The residualsolid was then dissolved in methanol (50 mL) and precipitated with ethylacetate (300 mL). The solid was again recrystallized from methanol-ethylacetate (15 mL/150 mL), washed with ethyl acetate (2×25 mL) and dried inan oven under high vacuum at 50° C. overnight. The yield was 4.21 g(39%). TLC (silica gel, 4:1 methylene chloride-methanol) R_(f)=0.34.

Preparation of 3-Methyl-2-(2-N-phenyl)ethenylbenzothiazolium Iodide (12a). To a solution of 2,3-dimethylbenzothiazolium bromide (11 a, 1.01 g)in ethanol (30 mL) was added excess of ethyl N-phenylformidiate (1.5 g)and stirred at room temperature for 4 days. Evaporated off solvent underreduced pressure to dryness. The solid was subjected to columnchromatography (silica gel, methylene chloride/methanol) purification.The fractions contained the product were combined and concentrated todryness under reduced pressure. The solid was dissolved in methanol (10mL) and precipitated with ethyl ether (200 mL). After drying in an ovenat 50° C. under high vacuum overnight there was obtained 0.88 g (60%) ofa yellow solid. TLC (silica gel, 4:1 methylene chloride-methanol)R_(f)=0.60.

Preparation of 3-(2-Hydroxy)ethyl-2-(2-N-phenyl)ethenylbenzothiazoliumBromide (12 b). To a solution of3-(2-hydroxy)ethyl-2-methylbenzothiazolium bromide (11 b, 1.19 g) in amixed solvent of methanol/ethanol (3:1, 80 mL) was added excess of ethylN-phenylformidiate (3.0 g) and stirred at room temperature for 36 h.Evaporated off solvent under reduced pressure to dryness. The solid wassubjected to column chromatography (silica gel, methylenechloride/methanol) purification. The fractions contained the productwere combined and concentrated to dryness under reduced pressure. Thesolid was dissolved in methanol (10 mL) and precipitated with ethylether (200 mL). After drying in an oven at 50° C. under high vacuumovernight there was obtained 1.04 g (63%) of a yellow solid. TLC (silicagel, 4:1 methylene chloride-methanol) R_(f)=0.58.

Preparation of Dye Compound designated as 13 under Synthesis Scheme IIin Example 1C

To a solution of 3-(2-hydroxy)ethyl-2-(2-N-phenyl)ethenylbenzothiazoliumbromide (12 b, 41.9 mg) and 1-benzyllepidinium bromide (6, 33.4 mg) inmethylene chloride (5 mL) was added triethylamine (44.3 μL) and aceticanhydride (30.1 μL). After stirring at room temperature for 1 hour thesolvent was evaporated off under reduced pressure to give a blue solid.Redissolved the solid in methanol (10 mL) and added ethyl acetate (200mL). The resultant solid was collected via filtration, washed with ethylacetate (3×10 mL) and dried. Further drying the solid in an oven at 50°C. under high vacuum overnight afforded 49.1 mg (89%) of a blue solid.TLC (silica gel, 9:1 methylene chloride-methanol) R_(f)=0.43. AbsorptionSpectrum: 636 nm (methanol).

Preparation of Dye Compound designated as 14 under Synthesis Scheme IIin Example 1C

To a solution of 3-(2-hydroxy)ethyl-2-(2-N-phenyl)ethenylbenzothiazoliumbromide (12 b, 120.5 mg) and 1-(3′-5′-dimethoxybenzyl) lepidiniumbromide (7, 119.5 mg) in methylene chloride (15 mL) and methanol (1 mL)was added triethylamine (133 μL) and acetic anhydride (90 μL). Afterstirring at room temperature for 30 min the solvent was evaporated offunder reduced pressure to give a blue solid. Redissolved the solid inmethanol (10 mL) and added acetone (100 mL) and ethyl acetate (100 mL).The resultant solid was collected via filtration, washed with ethylacetate (3×20 mL) and dried. Further drying the solid in an oven at 50°C. under high vacuum overnight afforded 147.3 mg (80%) of a blue solid.TLC (silica gel, 4:1 methylene chloride-methanol) R_(f)=0.91. AbsorptionSpectrum: 637 nm (methanol).

Preparation of Dye Compound designated as 15 under Synthesis Scheme IIin Example 1C

To a solution of 3-(2-hydroxy)ethyl-2-(2-N-phenyl)ethenylbenzothiazoliumbromide (12 b, 171.1 mg) and 1-(4′-carboxymethyl-benzyl)lepidiniumbromide (8, 168.8 mg) in methylene chloride (15 mL) and methanol (1 mL)was added triethylamine (189 μL) and acetic anhydride (128 μL). Afterstirring at room temperature for 1 h the solvent was evaporated offunder reduced pressure to give a blue solid. Redissolved the solid inmethanol (10 mL) and added acetone (50 mL) and ethyl acetate (50 mL).The resultant solid was collected via filtration, washed with ethylacetate (3×30 mL) and dried. Further drying the solid in an oven at 50°C. under high vacuum overnight afforded 208.9 mg (80%) of a blue solid.TLC (silica gel, 4:1 methylene chloride-methanol) R_(f)=0.80. AbsorptionSpectrum: 639 nm (methanol).

Preparation of Dye Compound designated as 16 under Synthesis Scheme IIin Example 1C

To a solution of 3-methyl-2-(2-N-phenyl)ethenylbenzothiazolium iodide(12 a, 144.6 mg) and 1-(2′-boronic acid)benzyllepidinium Bromide (9,131.3 mg) in methylene chloride (5 mL) and methanol (1 mL) was addedtriethylamine (153 μL) and acetic anhydride (104 μL). After stirring atroom temperature for 15 min the solvent was evaporated off under reducedpressure to give a blue solid. Redissolved the solid in methanol (5 mL)and added ethyl acetate (200 mL). The resultant solid was collected viafiltration, washed with ethyl acetate (3×30 mL) and dried. Furtherdrying the solid in an oven at 50° C. under high vacuum overnightafforded 107 mg (50%) of a blue solid. TLC (silica gel, 4:1 methylenechloride-methanol) R_(f)=0.58. Absorption Spectrum: 634 nm (methanol).

D. Dye Stock Solution

Stock solutions of dyes at 5 mM concentration in dimethylsulfoxide(DMSO) were prepared by dissolving a definite quantity of each dyecompound in appropriate volume of DMSO.

E. Dye Spectroscopy

For spectroscopic measurements, individual sample solution of each dyein PBS was prepared by diluting portions of its stock solution intophosphate-buffered saline (PBS). Specifically, in one experiment, thedye solutions were prepared by mixing 2 μl of the 5 mM stock dyesolution with 5 ml of PBS to give a final 2 μM dye concentration.Fluorescence spectra of the above solutions were measured with aspectrofluorometer using an excitation wavelength of 633 nm.

Solutions of the dyes of this invention bound to RNA were prepared bycombining 2 μl of the 5 mM stock solution of the dye and a 5 ml solutionof RNA dissolved in PBS at a RNA concentration of 1 mg/ml. Fluorescencespectrum of each Dye/RNA solution was measured with a spectrofluorometerusing an excitation wavelength of 633 nm.

Solutions of the dyes of this invention bound to DNA were prepared bycombining 2 μl of the 5 mM stock solution of the dye and a 5 ml solutionof DNA dissolved in PBS at a DNA concentration of 0.5 mg/ml Fluorescencespectrum of each Dye/DNA solution was measured with a spectrofluorometerusing an excitation wavelength of 633 nm.

F. Flow Cytometry For Red Excitable Dyes

An XL™ flow cytometer [Beckman Coulter Inc., Miami, Fla.] was used toconduct experiments described herein using the red-excitable dyes of theinvention. The flow cytometer was modified from a standard XL™ flowcytometer by incorporating a HeNe laser (632.8 nm) and two red-sensitivephotomultiplier tubes (PMT). Approximately 11.5 mW of 632.8 nm laserlight from the above laser was incident on the beam shaping optics ofthe flow cytometer. Forward light scatter (FS), side scatter (SS) andone fluorescence (FL) parameter in the orthogonal direction weremeasured to analyze the red cells. The red cells were gated on a SS vs.FS dotplot. A fixed gate was then used to enumerate retics on a FL vs.FS dotplot.

G. Reference Analyses

Separate analyses for reticulocytes were performed using either (1) thestandard Beckman Coulter reticulocyte reagents reticONE™, which is basedon a blue-excitable metachromatic fluorescent dye compound, employing astandard XL™ flow cytometer [Beckman Coulter Inc., Miami, Fla.] having a488 nm argon laser as the illumination source, or (2) the conventionalRetic-Count™ reticulocyte enumeration method (Becton Dickinson Cat.#349204) using Thiazole orange excited at 488 nm in a FACSCAN™ flowcytometer. In the reticONE™ method, FS, SS, and two FL parameters weremeasured to analyze the red cells stained with the blue-excitablefluorescent dye contained in the reticONE™ reagent. Out of the two FLparameters measured, one is measured at 525 nm, which corresponds tofluorescence due to the dye bound to DNA, and the other is measured at675 nm, which corresponds to the dye bound to RNA. An automated gatingalgorithm, available in commercial XL™ flow cytometers, calculatedreticulocyte percentage from a DNA vs. RNA fluorescence dotplot. Thismethod is described in U.S. Pat. No. 5,639,666.

For the Retic-Count™ method, FS, SS, and one FL parameter were measuredto analyze the red cells stained with the blue-excitable fluorescent dyeThiazole Orange contained in the Retic-Count™ reagent.

Example 2—Fluorescence Comparison of the Dyes of Present Invention inBound and Unbound States

In order to identify a fluorescent dye as a suitable candidate forreticulocyte enumeration, the fluorescence properties of severaldifferent compounds were determined. For reticulocyte enumeration, it ismost desirable to have dyes that are weakly fluorescent when they arenot bound to RNA, but show significant enhancement in fluorescenceintensity when they are bound to RNA. However, it is not possible topredict which dye compound will have this ideal combination effects onits fluorescence activity both in absence and in presence of RNA.Significantly, the inventors found that the dyes of the presentinvention had favorable fluorescence properties required for sensitivedetection of RNA.

For fluorescence measurements, a stock solution of each compound in DMSOwas made (as described in Example 1D). A portion of this stock solutionwas diluted in PBS to obtain a dye concentration of 2 μm. 1 ml of thisdilute solution was placed in a glass cuvette and the fluorescencespectra obtained exciting the dye at its excitation wavelength maximum.Next, 1 ml of a solution of free calf liver RNA (Sigma) dissolved in PBSat a concentration of 8 mg/ml, and a measured volume of the dye stocksolution was mixed to obtain a dye concentration of 2 μM in the RNAsolution. This dye solution diluted in the RNA solution was placed in aglass cuvette and the fluorescence spectra measured using the sameexcitation as in the previous measurement. As an example, FIG. 1A showsthe comparison of the fluorescence spectra of the dye compound ReticRed1in PBS solution with (see the upper curve) and without RNA (see thelower curve) present in the solution.

In FIG. 1B, comparison of fluorescence intensity of several compounds inpresence of RNA in solution are presented. In this FIG. 1B, thefluorescence intensities of the dyes are shown relative to thefluorescence intensity of a commercial dye Syto 62 (Molecular ProbesInc.) used herein as a reference standard. The structures of theadditional compounds included in this study and shown in FIG. 1B but notalready described above are shown in Table 2.

Table 1 compares the fluorescence intensities of the dyes of the presentinvention, ReticRed1 through ReticRed6 (concentration 2 μM), when theyare not bound to RNA (unbound to RNA) and when they are in presence ofRNA (bound to RNA). These intensities were obtained at the peak emissionwavelengths of each fluorescence spectra measured as described above.FIG. 1C compares the fluorescence intensities of the RNA-bound andunbound dyes in graphical form.

TABLE 1 Fluorescence Fluorescence Intensity enhancement ratio DYEUnbound to RNA Bound to RNA (Bound/Unbound) ReticRed1 2.49 791.23317.763 ReticRed2 1.72 643.77 374.285 ReticRed3 1.28 639.68 499.75 ReticRed4 1.72 713.4  414.767 ReticRed5 2.24 656.06 292.884 ReticRed62.75 619.2  225.164

TABLE 2 GS6262-43

GS6262-45

GS6262-55

GS6262-58

GS6262-60

GS6262-62

GS6262-82

GS6262-88

GS6262-07

GS6262-76

GS6262-74

GS6262-16

Example 3—Staining of Reticulocytes with ReticRed1 in Isotonic SalineSolution ISOFLOW™

A blood sample procured from a local hospital was used to determine thestaining kinetics of ReticRed1 (prepared as described in Examples 1A andD) alone. The blood sample was first analyzed by two independentreference automated methods: the reticONE™ method [described in Example1G] and the New Methylene Blue method, a commercial method that isavailable for use in Gen*S™ instruments (Beckman Coulter). The reticONE™reference method gave a retic percentage of 7% and the Gen*S™ methodgave a retic percentage of 6.7%.

For measurements using ReticRed1, 1 μl of whole blood was added to 1 mlof an isotonic saline solution containing a 5 μm ReticRed1 (diluted fromthe 5 mM stock solution described in Example 1C) solution and incubatedfor 10 and 40 minutes. The sample was then analyzed in a XL™ flowcytometer modified to incorporate a red HeNe laser (632.8 nm) asdescribed in Example 1E. When whole blood was treated with ReticRed1 andanalyzed in flow, red fluorescence in the 660 nm band resulted from apopulation of red cells, and indicated the presence of nucleic acidbound to ReticRed1 in such cells. This fluorescence is due toreticulocytes stained with ReticRed1. The flow cytometry data can besummarized according to incubation durations as follows: (a) 10 minutes:Total count 42660, Red Cells 32841, Reticulocytes 801, calculated reticpercentage 2.4% (b) 40 minutes: Total count: 46199, Red cells 39713,Reticulocytes 2310, calculated retic percentage 5.8%. See, FIGS. 2A and2B.

These results clearly demonstrate that the ReticRed1 dye alone requiresa relatively long period of time to effectively stain the intracellularRNA.

Example 4—Rapid Staining of Reticulocytes with ReticRed1 Using WholeBlood Samples

Although Example 3 above demonstrated that the dye ReticRed1 in anisotonic saline solution takes a relatively long time to stainreticulocytes, we found that using this dye to stain reticulocytes inpresence of additional reagent compositions of this invention can reducethe incubation time for the staining significantly. Runs using fourseparate blood samples are presented to demonstrate rapid staining ofreticulocytes by the dye ReticRed1.

A. Run 1

1 μl of whole blood (from the same donor whose blood was used to conductthe analyses described in example 3 above) was added to 1 ml of asolution comprising a sphering reagent that spheres the red cells, thedetergent Igepal (Sigma CA-630) at a concentration of 0.01%, the dyeReticRed1 at a concentration of 5 μM, and a solution ofp-toluenesulfonic acid monohydrate at a final concentration of 5 μM, andincubated for about 1 minute. The sample was then analyzed in a XL™ flowcytometer modified to incorporate a red HeNe laser (632.8 nm). Thesphering reagent is a solution comprising about 20 μg/mlDodecyl-β-D-maltoside and 0.05% Proclin 300 in PBS at about pH 7.4 andabout 290 mOsm.

The sample so stained with ReticRed1 was then analyzed in a flowcytometer using 632.8 nm excitation from a HeNe laser. Bright redfluorescence in the 660 nm band resulted from the reticulocytes. Theresults of this experiment are shown in FIG. 3. The distribution of thecells in this dotplot, showing forward scatter versus fluorescence, isconsistent with the distribution of mature red cells and reticulocytespreviously shown by Tanke et. al. [cited above], in relation to theirwork on fluorescence based reticulocyte measurements. The percentage ofreticulocytes enumerated by the present method was 7.3%.

B. Run 2

1 μl of whole blood from another abnormal donor was added to 1 ml of asolution comprising a sphering reagent (described in Example 4A, Run 1above) that spheres the red cells, the detergent Igepal (Sigma CA-630)at a concentration of 0.01%, the dye ReticRed1 at a concentration of 5μm, and a solution of p-toluenesulfonic acid monohydrate at a finalconcentration of 5 μM, and incubated for about 1 minute. The sample wasthen analyzed in a XL™ flow cytometer modified to incorporate a red HeNelaser (632.8 nm).

The sample so stained with the dye ReticRed1 was then analyzed in a flowcytometer using 632.8 nm excitation from a HeNe laser. The result ofthis experiment is shown in FIG. 4. The distribution of the cells inthis forward scatter versus fluorescence dotplot clearly shows thereticulocytes with high fluorescence intensity. The percentage ofreticulocytes enumerated by this experiment was 7.5%. An independentreference value for reticulocyte percentage in this sample was 8%.

C. Run 3

1 μl of whole blood from a normal donor was added to 1 ml of a solutioncomprising a sphering reagent (described in Example 4A, Run 1 above)that spheres red cells, detergent Igepal (Sigma CA-630) at aconcentration of 0.01%, the dye ReticRed1 at a concentration of 5 μM,and a solution of p-toluenesulfonic acid monohydrate at a finalconcentration of 5 μM, and incubated for about one minute. The samplewas then analyzed in a XL™ flow cytometer modified to incorporate a redHeNe laser (632.8 nm). The blood sample so stained with the dyeReticRed1 was analyzed in flow using 632.8 nm excitation from a HeNelaser. The result of this experiment is shown in FIG. 5. Thereticulocyte percentage enumerated by this experiment was 0.82%. Anindependent reference measurement of the blood from the same donor usingnew methylene blue in a commercially available clinical hematologyanalyzer gave retic percentage of 0.95%.

D. Run 4

1 μl of abnormal whole blood from a donor was added to 1 ml of asolution comprising a sphering reagent (described in Example 4A, Run 1above) that spheres red cells, detergent Igepal (Sigma CA-630) at aconcentration of 0.01%, the dye ReticRed1 at a concentration of 5 μM,and a solution of sodium p-toluenesulfonate at a final concentration of5 μM, and incubated for about one minute. The sample was then analyzedin a XL™ flow cytometer modified to incorporate a red HeNe laser (632.8nm).

The sample blood so stained with ReticRed1 was then analyzed in flowcytometer using 632.8 nm excitation from a HeNe laser. The result ofthis experiment is shown in FIG. 6. The reticulocyte percentageenumerated by this experiment was 13.7%. An independent reference methodfor this same sample gave a retic percentage value of 13%.

Example 5—Rapid Staining of Reticulocytes with ReticRed3 Using WholeBlood Samples

A. Run 1

2 μl of whole blood from an abnormal donor having a high reticulocytecount was added to 1 ml of a solution comprising a sphering reagent(described in Example 4A, Run 1 above) that spheres the red cells, thedetergent Igepal® (Sigma CA-630) at a concentration of 0.01%, the dyeReticRed1 at a concentration of 5 μM, and a solution of sodiump-toluenesulfonate at a final concentration of 50 μM, and was gentlymixed/incubated for about a second. The mixture was then aspirated intothe XL™ flow cytometer containing the red HeNe laser for analysis.

The result of this experiment is shown in FIG. 7, where thereticulocytes are distinguished from the mature red blood cells by thehigher fluorescence intensities associated with them. The percentage ofreticulocytes enumerated by this experiment was 13.8%. An independentreference value for reticulocyte percentage measured for this sampleusing the conventional Retic-Count™ reticulocyte enumeration method(using Thiazole orange excited at 488 nm) in a FACSCAN flow cytometerwas 13.3%.

B. Run 2

2 μl of whole blood from an abnormal donor having low reticulocytecounts was added to 1 ml of a solution comprising a sphering reagent(described in Example 4A, Run 1 above) that spheres the red cells, thedetergent Igepal (Sigma CA-630) at a concentration of 0.01%, the dyeReticRed1 at a concentration of 5 μM, and a solution of sodiump-toluenesulfonate at a final concentration of 50 μM, and was gentlymixed/incubated for about a second. The mixture was then aspirated intothe XL™ flow cytometer containing the red HeNe laser for analysis.

The result of this experiment is shown in FIG. 8. The percentage ofreticulocytes enumerated by this experiment was 1%. An independentreference value for reticulocyte percentage measured for this sampleusing the conventional Retic-Count™ reticulocyte enumeration method(Thiazole orange excited at 488 nm) in a FACSCAN flow cytometer was1.3%.

Example 6—Comparison of Fluorescence Intensities the Dyes of PresentInvention in Bound and Unbound States

To compare the relative fluorescence intensities of the dyes in boundand unbound states, fluorescence measurements were conducted in aspectrofluorometer by exciting each dye solution at 633 nm underappropriate conditions, recording the fluorescence spectrum of eachsample, and measuring the intensities at the peak of these fluorescencespectra.

To measure the fluorescence intensities of the dye solution in absenceof DNA or RNA (i.e., unbound state), first a stock solution of eachcompound in DMSO was prepared (as described in Example 1D). A portion ofthe stock solution for the Dye-1 was diluted in PBS to obtain a dyeconcentration of 2 μl. Next, 1 ml of this dilute solution was placed ina glass cuvette and fluorescence spectrum of this solution was measuredby a spectrofluorometer using an excitation wavelength of 633 nm.

Next, 1 ml of a solution of free Torula yeast RNA (Sigma) dissolved inPBS at a concentration of 1 mg/ml, and a measured volume of the dyestock solution of the Dye-1 was mixed to obtain a dye concentration of 2μM in the RNA solution. The fluorescence spectrum of this dye solutionin presence of RNA was measured by illuminating the sample at 633 nm.FIG. 9A shows the comparison of the fluorescence spectra of the dyeDye-1 in PBS solution with (see the upper curve) and without RNA (seethe lower curve) present in the solution.

For fluorescence measurements in DNA, 1 ml of a solution of free Calfthymus DNA (Sigma) dissolved in PBS at a concentration of 0.5 mg/ml, anda measured volume of the dye stock solution was mixed to obtain a dyeconcentration of 2 μM in the RNA solution. Next, the fluorescencespectrum of the dye solution was measured by illuminating the sample at633 nm. FIG. 9B shows the comparison of the fluorescence spectra of thedye Dye-1 in PBS solution with (see the upper curve) and without DNA(see the lower curve) present in the solution.

In the same manner, the experiments described above were repeated forthe dye Dye-2 through Dye-6 (see FIGS. 9C- and 9L).

Table 3 compares the relative fluorescence intensities of the dyes ofthe present invention, Dye-1 and Dye-2 (concentration 2 μM), when theyare not bound to RNA (unbound to RNA), when they are in presence of RNA(bound to RNA), and when they are in presence of DNA (bound to DNA).These intensities were obtained at the peak of the respectivefluorescence spectra as described above.

TABLE 3 Fluorescence Intensity DYE Bound to RNA Bound to DNA (conc. 2μm) Unbound to RNA 1 mg/ml 0.5 mg/ml Dye-1 10 825 578 Dye-2 18 560 760

Table 4 compares the fluorescence intensities of the Group-III dyes(Dye-3 through Dye-6) of the present invention, when they are not boundto RNA (unbound to RNA) and when they are in presence of RNA (bound toRNA). The intensities were measured at the peak of the emission spectra.

Table-5 compares the fluorescence intensities of the Group-III dyes(Dye-3 through Dye-6) of the present invention, when they are not boundto DNA (unbound to DNA) and when they are in presence of DNA (bound toDNA). The intensities were measured at the peak of the emission spectra.

TABLE 4 Fluorescence Intensity DYE Bound to RNA (conc. 2 μm) Unbound toRNA 1 mg/ml Dye-3 10 328 Dye-4 12 370 Dye-5 10 420 Dye-6  6 410

TABLE 5 Fluorescence Intensity Bound to DNA DYE Unbound to DNA 0.5 mg/mlDye-3 15 400 Dye-4 12 440 Dye-5 10 660 Dye-6  4 380

Example 7—Rapid Staining of Reticulocytes with Dye-1 Using Whole BloodSamples

We found that the dye Dye-1 can pass through the cell membrane ofreticulocytes rapidly in presence of the reagent compositions of thisinvention, thus reducing the incubation time for the stainingsignificantly. Experiments using three separate blood samples arepresented to demonstrate rapid staining of reticulocytes by the dyeDye-1. We refer to these three experiments as Run 1, Run 2 and Run 3respectively.

A. Run 1

1 μl of whole blood from a normal donor was added to 1 ml of a solutioncomprising a sphering reagent that spheres the red cells, the detergentIgepal (Sigma CA-630) at a concentration of 0.01%, the dye Dye-1 at aconcentration of 5 μM, and aspirated, immediately thereafter, into anXL™ flow cytometer, modified to incorporate a red HeNe laser (632.8 nm),for analysis. The sphering reagent is a solution comprising about 20μg/ml Dodecyl-β-D-maltoside and 0.05% Proclin 300 in PBS at about pH 7.4and about 290 mOsm.

FIG. 10A. shows the fluorescence from reticulocytes relative to themature red blood cells for this sample. The percentage of reticulocytesenumerated by the present method was 1.9%, which was within the expectednormal range.

B. Run 2

1 μl of whole blood from an abnormal donor was added to 1 ml of asolution comprising a sphering reagent (described in Example 7, Run 1above) that spheres the red cells, the detergent Igepal (Sigma CA-630)at a concentration of 0.01%, and the dye Dye-1 at a concentration of 5μM. Immediately thereafter, the mixture was aspirated into an XL™ flowcytometer modified to incorporate a red HeNe laser (632.8 nm), foranalysis.

FIG. 10B shows the fluorescence from reticulocytes relative to themature red blood cells for this sample. The distribution of the cells inthis forward scatter versus fluorescence dotplot clearly shows thereticulocytes with relatively high fluorescence intensity. Thepercentage of reticulocytes enumerated by this experiment was 10.6% Anindependent reference value for reticulocyte percentage in this samplewas 12%.

C. Run 3

1 μl of whole blood from another abnormal donor was added to 1 ml of asolution comprising a sphering reagent (described in Example 7, Run 1above) that spheres the red cells, the detergent Igepal (Sigma CA-630)at a concentration of 0.01%, and the dye Dye-1 at a concentration of 5μM. Immediately thereafter, the mixture was aspirated into an XL™ flowcytometer, modified to incorporate a red HeNe laser (632.8 nm), foranalysis.

FIG. 10C shows the fluorescence from reticulocytes relative to themature red blood cells for this sample. The distribution of the cells inthis forward scatter versus fluorescence dotplot clearly shows thereticulocytes with high fluorescence intensity. The percentage ofreticulocytes enumerated by this experiment was 26.3%. An independentreference value for reticulocyte percentage in this sample was 27%.

Example 8—Rapid Staining of Reticulocytes with Dye-2 Using Whole BloodSamples

We found that Dye-2 can also rapidly pass through the cell membrane ofreticulocytes in presence of the reagent compositions of this invention,thus reducing the incubation time for the staining significantly.Experiments using three different blood samples are presented todemonstrate rapid staining of reticulocytes by the dye Dye-2. We referto these three experiments as Run 4, Run 5 and Run 6 respectively.

A. Run 4

1 μl of whole blood from a normal donor was added to 1 ml of a solutioncomprising a sphering reagent that spheres the red cells, the detergentIgepal (Sigma CA-630) at a concentration of 0.01%, the dye Dye-2 at aconcentration of 5 μM, and aspirated, immediately thereafter, into anXL™ flow cytometer, modified to incorporate a red HeNe laser (632.8 nm),for analysis. The sphering reagent is a solution comprising about 20μg/ml Dodecyl-β-D-maltoside and 0.05% Proclin 300 in PBS at about pH 7.4and about 290 mOsm.

FIG. 11A shows the fluorescence from reticulocytes relative to themature red blood cells for this sample. The percentage of reticulocytesenumerated by the present method was 1.8%.

B. Run 5

1 μl of whole blood from an abnormal donor was added to 1 ml of asolution comprising a sphering reagent (described in Example 7, Run 1above) that spheres the red cells, the detergent Igepal (Sigma CA-630)at a concentration of 0.01%, and the dye Dye-2 at a concentration of 5μM. Immediately thereafter, the mixture was aspirated into an XL™ flowcytometer, modified to incorporate a red HeNe laser (632.8 nm), foranalysis.

FIG. 11B shows the fluorescence from reticulocytes relative to themature red blood cells for this sample. The distribution of the cells inthis forward scatter versus fluorescence dotplot clearly shows thereticulocytes with high fluorescence intensity. The percentage ofreticulocytes enumerated by this experiment was 12.5%. An independentreference value for reticulocyte percentage in this sample was 12%.

C. Run 6

1 μl of whole blood from a high retic abnormal donor was added to 1 mlof a solution comprising a sphering reagent (described in Example 7, Run1 above) that spheres the red cells, the detergent Igepal (Sigma CA-630)at a concentration of 0.01%, and the dye Dye-2 at a concentration of 5μM. Immediately thereafter, the mixture was aspirated into an XL™ flowcytometer, modified to incorporate a red HeNe laser (632.8 nm), foranalysis.

FIG. 11C shows the fluorescence from reticulocytes relative to themature red blood cells for this sample. The distribution of the cells inthis forward scatter versus fluorescence dotplot clearly shows thereticulocytes with high fluorescence intensity. The percentage ofreticulocytes enumerated by this experiment was 24%. An independentreference value for reticulocyte percentage in this sample was 27%.

Example 9—Rapid Staining of Reticulocytes with Dye-3 Using Whole BloodSamples

A. Run 7

1 μl of whole blood from a normal donor was added to 1 ml of a solutioncomprising a sphering reagent that spheres the red cells, the detergentIgepal (Sigma CA-630) at a concentration of 0.01%, the dye Dye-3 at aconcentration of 5 μM, and aspirated, immediately thereafter, into anXL™ flow cytometer, modified to incorporate a red HeNe laser (632.8 nm),for analysis. The sphering reagent is a solution comprising about 20μg/ml Dodecyl-β-D-maltoside and 0.05% Proclin® 300 in PBS at about pH7.4 and about 290 mOsm. The percentage of reticulocytes enumerated bythe present method was 2.3% (see FIG. 4A).

B. Run 8

1 μl of whole blood from an abnormal donor was added to 1 ml of asolution comprising a sphering reagent (described in Example 7, Run 1above) that spheres the red cells, the detergent Igepal (Sigma CA-630)at a concentration of 0.01%, and the dye Dye-3 at a concentration of 5μM. Immediately thereafter, the mixture was aspirated into an XL™ flowcytometer, modified to incorporate a red HeNe laser (632.8 nm), foranalysis. The percentage of reticulocytes enumerated by this experimentwas approximately 14.6% (see FIG. 4A). An independent reference valuefor reticulocyte percentage in this sample was 12%.

C. Run 9

1 μl of whole blood from another abnormal donor was added to 1 ml of asolution comprising a sphering reagent (described in Example 7, Run 1above) that spheres the red cells, the detergent Igepal (Sigma CA-630)at a concentration of 0.01%, and the dye Dye-3 at a concentration of 5μM. Immediately thereafter the mixture was aspirated into an XL™ flowcytometer, modified to incorporate a red HeNe laser (632.8 nm), foranalysis. The percentage of reticulocytes enumerated by this experimentwas approximately 24% (see FIG. 12A). An independent reference value forreticulocyte percentage in this sample was 27%.

All publications cited in this specification are incorporated herein byreference herein. While the invention has been described with referenceto a particularly preferred embodiment, it will be appreciated thatmodifications can be made without departing from the spirit of theinvention. Such modifications are intended to fall within the scope ofthe appended claims.

What is claimed is:
 1. A dye having the formula:

wherein n is 0, 1, 2, or 3; R1 is H, alkyl, or an alkoxy group; R2 isCH₂(CH₂)_(m)OAc, wherein m is 0, 1, 2, or 3; X is O, S, or C(CH₃)₂; R isCH₃, CH(CH₃)₂, CH₂CH₂OAc, alkyl, alkylsulfonate, or hydroxyalkyl and B−is a counteranion.
 2. The dye according to claim 1, wherein n 1; R₁=H;R=CH₃, R2=CH₂CH₂OAc; X=S, and B⁻ is Br⁻.
 3. The dye according to claim1, wherein n=1, R1=H, R2=R=CH₂CH₂OAc, X=S, and B⁻ is Br⁻.
 4. A dyecomposition for staining blood cells comprising the compound of claim 1,wherein the blood cells are nucleated red blood cells.
 5. A dyecomposition for staining blood cells comprising the compound of claim 1,wherein the blood cells are reticulocytes.
 6. The dye for stainingreticulocytes according to claim 5, further comprising a surfactant. 7.The dye according to claim 1, further comprising a detergent in anamount of about 0% up to about 1% (w/v).
 8. The dye according to claim1, further comprising a sulfonic acid or a salt thereof.
 9. A method forstaining nucleic acid comprising the step of contacting a blood cellsample containing nucleic acid with the dye according to claim
 1. 10.The method according to claim 9, wherein said blood cell sample iscontacted by the dye in the presence of a surfactant.
 11. The methodaccording to claim 10, wherein said blood cell sample is contacted bythe dye after lysing mature red blood cells and reticulocytes, butpreserving nucleated red blood cells in the blood cells sample.
 12. Themethod according to claim 11, wherein a mixture of the blood cell sampleand dye is incubated for up to about one minute.
 13. A method foranalysis of reticulocytes comprising the steps of: (a) contacting ablood cell sample containing reticulocytes with the dye according toclaim 1 such that the reticulocytes are stained by the dye; and (b)analyzing the stained reticulocytes by flow cytometry to detect thepresence of reticulocytes.
 14. The method according to claim 13, whereinsaid analysis by flow cytometry includes measurement of one fluorescenceparameter and at least one parameter selected from the group consistingof light scatter, axial light loss, DC electrical impedance, and radiofrequency (RF) conductivity and combinations thereof.
 15. A dyeaccording to the formula:

wherein n is 0, 1, or 2; R1 is H, alkyl, or an alkoxy group; R2 isCH₂(CH₂)_(m)OH, or CH₃ and wherein m is 0, 1, 2, or 3; X is O, S, orC(CH₃)₂; B⁻ is a counter anion, and R3=OCH₃ or H; R4=COOCH₃ or H;R5=OCH₃ or H; and R6=H or B(OH)₂.
 16. The dye according to claim 15,wherein n=1; X=S; R2=CH₂CH₂OH; R1=R4=R6=H; R3=R5=OCH₃ and B⁻ is Br⁻. 17.The dye according to claim 15, wherein n=1; X=S; R1=R3=R5=R6=H;R2=CH₂CH₂OH; R4=COO.CH₃ and B⁻ is Br⁻.
 18. The dye according to claim15, wherein n=1; X=S; R₁=R₃=R₄=R₅=R₆=H; R₂=CH₂CH₂OH and B⁻ is Br⁻. 19.The dye according to claim 15, wherein n=1; X=S; R₁=R₆=R₄=R₅=H;R2=CH₂CH₂OH; R₆=B(OH)₂ and B⁻ is I⁻.
 20. A dye composition for stainingblood cells comprising the compound of claim 15, wherein the blood cellsare nucleated red blood cells.
 21. A dye composition for staining bloodcells comprising the compound of claim 15, wherein the blood cells arereticulocytes.
 22. The dye for staining reticulocytes according to claim21, further comprising a surfactant.
 23. The dye according to claim 15,further comprising a detergent in an amount of about 0% up to about 1%(w/v).
 24. The dye according to claim 15, further comprising a sulfonicacid or a salt thereof.
 25. A method for staining nucleic acidcomprising the step of contacting a blood cell sample containing nucleicacid with the dye according to claim
 15. 26. The method according toclaim 25, wherein said blood cell sample is contacted by the dye in thepresence of a surfactant.
 27. The method according to claim 26, whereinsaid blood cell sample is contacted by the dye after lysing mature redblood cells and reticulocytes, but preserving nucleated red blood cellsin the blood cells sample.
 28. The method according to claim 27, whereina mixture of the blood cell sample and dye is incubated for up to aboutone minute.
 29. A method for analysis of reticulocytes comprising thesteps of: (a) contacting a blood cell sample containing reticulocyteswith the dye according to claim 15, such that the reticulocytes arestained by the dye; and (b) analyzing the stained reticulocytes by flowcytometry to detect the presence of reticulocytes.
 30. The methodaccording to claim 29, wherein said analysis by flow cytometry includesmeasurement of one fluorescence parameter and at least one parameterselected from the group consisting of light scatter, axial light loss,DC electrical impedance, and radio frequency (RF) conductivity andcombinations thereof.